Detailed information on the intramolecular interactions of protein amino groups has been largely unobtainable except to a limited extent from crystallographic studies. Reductive methylation with NaCNBH3 and (13C) formaldehyde introduces a specific probe for 13C nuclear magnetic resonance (NMR) investigation of protein amino groups (Jentoft and Dearborn, J. Biol Chem., 254: 4359-4365 (1979); Jentoft, et al., J. Biol. Chem., 254: 4366-4370 (1979). We have shown that the chemical shifts of 13C-enriched dimethylamino groups are sensitive to the environment and degree of protonation of the amino group, and have demonstrated that the pK of the dimethylamino groups are very similar to those of unmodified amino groups. The active site residue lysine 41 has been studied in methylated ribonuclease A. In the absence of active site ligands, the titration behavior of lysine 41 is biphasic with pK values of 5.7 and 9.0. The pK at 5.7 is likely to be due to a neighboring residue, probably histidine 12, which upon titration affects the environment of lysine 41. Binding studies have been performed with 12 different active site ligands, the majority of which cause perturbation of titration behavior of lysine 41; however, this is not the case with 2'-deoxycytidine. Methylated HEW Lysozyme was studied to determine if salt bridges and hydrogen bonds could form and be detected in methylated amino groups. Six of the seven lysozyme amino groups are involved in intramolecular interactions in the crystal, and our results suggest that, in solution, methylated amino groups maintain these interactios. One lysyl residue is highly perturbed: the chemical shift is at an abnormally high field over the pH range 3.5-9 and titrates toward normal values at both extremes of pH. The inflection centered at pH 2.5 suggests an interaction with a carboxyl residue and thus the titration behavior is interpreted as indicative of a salt bridge within the protein, consistent with crystallographic studies of lysozyme. These stdies are currently being extended to human hemoglobin, cytochrome C, and chymotrypsin.